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Regulation of p53 expression, phosphorylation and subcellular localization by a G-protein-coupled receptor.

Solyakov L, Sayan E, Riley J, Pointon A, Tobin AB - Oncogene (2009)

Bottom Line: In this study we show that a classical G(q/11)-coupled GPCR, the M(3)-muscarinic receptor, was able to regulate apoptosis through receptors that are endogenously expressed in the human neuroblastoma cell line, SH-SY5Y, and when ectopically expressed in Chinese hamster ovary (CHO) cells.This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53.This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, subcellular trafficking and modification of p53 in a manner that is, in part, dependent on the cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

ABSTRACT
G-protein-coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This superfamily of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In this study we show that a classical G(q/11)-coupled GPCR, the M(3)-muscarinic receptor, was able to regulate apoptosis through receptors that are endogenously expressed in the human neuroblastoma cell line, SH-SY5Y, and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M(3)-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, subcellular trafficking and modification of p53 in a manner that is, in part, dependent on the cell type.

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p53-mediates the apoptotic response to etoposide in SH-SY5Y but M3-muscarinic receptors protect against apoptosis independently of changes in p53 expression(A) SH-SY5Y cells treated with etoposide (Eto, 25μM) in the absence or presence of methylcholine (Met, 100μM) were lysed and the lysates processed for p53 expression. (B) SH-SY5Y cells were transfected with either siRNA duplex targeted to p53 or control duplex. 48 Hours later cells were treated for four hours with etoposide (Eto, 25mM) in the presence or absence of methacholine (Met 100μM). Cell Lysates were then prepared and probed for p53, PARP cleavage and caspase 3 processing. Shown is a typical experiment of at least three independent experiments.
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Figure 3: p53-mediates the apoptotic response to etoposide in SH-SY5Y but M3-muscarinic receptors protect against apoptosis independently of changes in p53 expression(A) SH-SY5Y cells treated with etoposide (Eto, 25μM) in the absence or presence of methylcholine (Met, 100μM) were lysed and the lysates processed for p53 expression. (B) SH-SY5Y cells were transfected with either siRNA duplex targeted to p53 or control duplex. 48 Hours later cells were treated for four hours with etoposide (Eto, 25mM) in the presence or absence of methacholine (Met 100μM). Cell Lysates were then prepared and probed for p53, PARP cleavage and caspase 3 processing. Shown is a typical experiment of at least three independent experiments.

Mentions: In light of the ability of the M3-muscarinic receptor to regulate the expression levels of p53 following a challenge with etoposide in CHO-m3 cells we tested if muscarinic receptor stimulation could change the expression levels of p53 in SH-SY5Y cells. Etoposide treatment of SH-SY5Y cells for two, four and eight hours resulted in a substantial increase in p53 expression that was not significantly affected by co-stimulation with methylcholine (Fig 3A). Hence, in contrast to CHO-m3 cells, M3-muscarinic receptors do not appear to regulate the expression levels of p53 in SH-SY5Y cells.


Regulation of p53 expression, phosphorylation and subcellular localization by a G-protein-coupled receptor.

Solyakov L, Sayan E, Riley J, Pointon A, Tobin AB - Oncogene (2009)

p53-mediates the apoptotic response to etoposide in SH-SY5Y but M3-muscarinic receptors protect against apoptosis independently of changes in p53 expression(A) SH-SY5Y cells treated with etoposide (Eto, 25μM) in the absence or presence of methylcholine (Met, 100μM) were lysed and the lysates processed for p53 expression. (B) SH-SY5Y cells were transfected with either siRNA duplex targeted to p53 or control duplex. 48 Hours later cells were treated for four hours with etoposide (Eto, 25mM) in the presence or absence of methacholine (Met 100μM). Cell Lysates were then prepared and probed for p53, PARP cleavage and caspase 3 processing. Shown is a typical experiment of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875175&req=5

Figure 3: p53-mediates the apoptotic response to etoposide in SH-SY5Y but M3-muscarinic receptors protect against apoptosis independently of changes in p53 expression(A) SH-SY5Y cells treated with etoposide (Eto, 25μM) in the absence or presence of methylcholine (Met, 100μM) were lysed and the lysates processed for p53 expression. (B) SH-SY5Y cells were transfected with either siRNA duplex targeted to p53 or control duplex. 48 Hours later cells were treated for four hours with etoposide (Eto, 25mM) in the presence or absence of methacholine (Met 100μM). Cell Lysates were then prepared and probed for p53, PARP cleavage and caspase 3 processing. Shown is a typical experiment of at least three independent experiments.
Mentions: In light of the ability of the M3-muscarinic receptor to regulate the expression levels of p53 following a challenge with etoposide in CHO-m3 cells we tested if muscarinic receptor stimulation could change the expression levels of p53 in SH-SY5Y cells. Etoposide treatment of SH-SY5Y cells for two, four and eight hours resulted in a substantial increase in p53 expression that was not significantly affected by co-stimulation with methylcholine (Fig 3A). Hence, in contrast to CHO-m3 cells, M3-muscarinic receptors do not appear to regulate the expression levels of p53 in SH-SY5Y cells.

Bottom Line: In this study we show that a classical G(q/11)-coupled GPCR, the M(3)-muscarinic receptor, was able to regulate apoptosis through receptors that are endogenously expressed in the human neuroblastoma cell line, SH-SY5Y, and when ectopically expressed in Chinese hamster ovary (CHO) cells.This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53.This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, subcellular trafficking and modification of p53 in a manner that is, in part, dependent on the cell type.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

ABSTRACT
G-protein-coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This superfamily of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In this study we show that a classical G(q/11)-coupled GPCR, the M(3)-muscarinic receptor, was able to regulate apoptosis through receptors that are endogenously expressed in the human neuroblastoma cell line, SH-SY5Y, and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M(3)-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, subcellular trafficking and modification of p53 in a manner that is, in part, dependent on the cell type.

Show MeSH
Related in: MedlinePlus